Modifying enzyme properties by site-directed mutagenesis has been limited to natural amino acid replacements, although molecular biological strategies for overcoming this restriction have recently been derived (Cornish, V. W. et al. (1995) Angew. Chem., Int. Ed. Engl. 34:621). However, the latter procedures are not generally easy to apply in most laboratories. In contrast, controlled chemical modification of enzymes offers broad potential for facile and flexible modification of enzyme structure, thereby opening up extensive possibilities for controlled tailoring of enzyme specificity.
Changing enzyme properties by chemical modification has been explored previously, with the first report being in 1966 by the groups of Bender (Polgar, L. et al. (1966) J. Am. Chem. Soc. 88:3153) and Koshland (Neet, K. E. et al. (1966) Proc. Natl. Acad. Sci. USA 56:1606), who created a thiolsubtilisin by chemical transformation (CH.sub.2 OH.fwdarw.CH.sub.2 SH) of the active site serine residue of subtilisin BPN' to cysteine. Interest in chemically produced artificial enzymes, including some with synthetic potential, was renewed by Wu, Z. -P. et al. (1989) J. Am. Chem. Soc. 111:4514; Bell, I. M. et al. (1993) Biochemistry 32:3754 and Peterson, E. B. et al. (1995) Biochemistry 34:6616, and more recently by Suckling, C. J. et al. (1993) Bioorg. Med. Chem. Lett. 3:531.
Enzymes are now widely accepted as useful catalysts in organic synthesis. However, natural, wild-type, enzymes can never hope to accept all structures of synthetic chemical interest, nor always to transform them stereospecifically into the desired enantiomerically pure materials needed for synthesis. This potential limitation on the synthetic applicabilities of enzymes has been recognized, and some progress has been made in to altering their specificities in a controlled manner using the site-directed and random mutagenesis techniques of protein engineering. However, modifying enzyme properties by protein engineering is limited to making natural amino acid replacements, and molecular biological methods devised to overcome this restriction are not readily amenable to routine application or large scale synthesis. The generation of new specificities or activities obtained by chemical modification of enzymes has intrigued chemists for many years, and continues to do so. The inventors have adopted the combined site-directed mutagenesis-chemical modification strategy since it offers virtually unlimited possibilities for creating new structural environments at any amino acid location.
U.S. Pat. No. 5,208,158 describes chemically modified detergent enzymes wherein one or more methionines have been mutated into cysteines. The cysteines are subsequently modified in order to confer upon the enzyme improved stability towards oxidative agents. The claimed chemical modification is the replacement of the thiol hydrogen with a C.sub.1-6 alkyl.
Although U.S. Pat. No. 5,208,158 has described altering the oxidative stability of an enzyme, it would also be desirable to develop one or more enzymes with altered properties such as activity, nucleophile specificity, substrate specificity, stereoselectivity, thermal stability, pH activity profile and surface binding properties for use in, for example, detergents or organic synthesis.